State Key Laboratory of Agrobiotechnology and MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
Curr Genet. 2020 Aug;66(4):835-847. doi: 10.1007/s00294-020-01063-1. Epub 2020 Mar 9.
Neurospora crassa is an excellent model fungus for studies on molecular genetics, biochemistry, physiology, and molecular cell biology. Along with the rapid progress of Neurospora research, new tools facilitating more efficient and accurate genetic analysis are in high demand. Here, we tested whether the dominant selective makers widely used in yeasts are applicable in N. crassa. Among them, we found that the strains of N. crassa are sensitive to the aminoglycoside antibiotics, G418 and nourseothricin. 1000 μg/mL of G418 or 50 μg/mL of nourseothricin is sufficient to inhibit Neurospora growth completely. When the neomycin phosphotransferase gene (neo) used in mammalian cells is expressed, N. crassa shows potent resistance to G418. This establishes G418-resistant marker as a dominant selectable marker to use in N. crassa. Similarly, when the nourseothricin acetyltransferase gene (nat) from Streptomyces noursei is induced by qa-2 promoter in the presence of quinic acid (QA), N. crassa shows potent resistance to nourseothricin. When nat is constitutively expressed by full-length or truncated versions of the promoter from the N. crassa cfp gene (NCU02193), or by the trpC promoter of Aspergillus nidulans, the growth of N. crassa in the presence of nourseothricin is proportional to the expression levels of Nat. Finally, these two markers are used to knock-out wc-2 or al-1 gene from the N. crassa genome. The successful development of these two markers in this study expands the toolbox for N. crassa and very likely for other filamentous fungi as well.
粗糙脉孢菌是研究分子遗传学、生物化学、生理学和分子细胞生物学的优秀模式真菌。随着脉孢菌研究的快速发展，需要新的工具来促进更高效和准确的遗传分析。在这里，我们测试了在酵母中广泛使用的显性选择性标记是否适用于粗糙脉孢菌。其中，我们发现粗糙脉孢菌菌株对氨基糖苷类抗生素新霉素和潮霉素敏感。1000μg/mL 的新霉素或 50μg/mL 的潮霉素足以完全抑制粗糙脉孢菌的生长。当哺乳动物细胞中的新霉素磷酸转移酶基因（neo）表达时，粗糙脉孢菌对新霉素表现出强烈的抗性。这确立了新霉素抗性标记作为在粗糙脉孢菌中使用的显性可选择标记。类似地，当 qa-2 启动子在奎尼酸（QA）存在下诱导来自链霉菌诺尔斯氏菌的潮霉素乙酰转移酶基因（nat）时，粗糙脉孢菌对潮霉素表现出强烈的抗性。当 nat 由粗糙脉孢菌 cfp 基因（NCU02193）的全长或截短版本的启动子或aspergillus nidulans 的 trpC 启动子组成型表达时，粗糙脉孢菌在潮霉素存在下的生长与 Nat 的表达水平成正比。最后，这两个标记被用于敲除粗糙脉孢菌基因组中的 wc-2 或 al-1 基因。本研究中这两个标记的成功开发扩展了粗糙脉孢菌的工具包，很可能也扩展了其他丝状真菌的工具包。
